Renal dehydropeptidase (DHP-I) (also known as dihydropeptidase I, microsomal dipeptidase, or EC 3.4.13.19) is a glycoprotein involved in hydrolysis of the peptide bond of dipeptides (Adachi 1990, Campbell 1966) which is mainly located in the brush border of the proximal tubular cells of the kidney. DPH-I is a homodimer with two subunits (α, β), each consisting of a 369-amino acid peptide (42 KDa). The subunits have four potential glycosilation sites, and a highly glycosilated form of 63 KDa may be obtained. The active site of each of the subunits forming the dimer (α, β) consists of zinc ions which are oriented towards the microvilli of the renal tubule (Nitanai 2002). DHP-I is involved in renal metabolism of gluthatione and in conversion of leukotriene D4 into leukotriene E4 (Kozak 1982) and is to date the only mammalian enzyme able to hydrolyze the beta-lactam ring (Campbell 1984). DHP-I is responsible for hydrolyzing the β-lactam ring of imipenem, inactivating it (Kaham 1983), but does not affect penicillins or cefalosporins.
DHP-I is anchored to the cell membrane by a covalent bond to a glycosyl-phosphatidyl-inositol (GPI) structure (Adachi 1990). Modification of the cell fate of the protein by the GPI anchor during protein translation is a membrane binding modality of more than 200 proteins in eukaryotic cells. In addition to allowing for protein binding to the membrane, it has important roles, particularly in signal translation or in the recognition process (Nosjean 1997). GPI residues are typically located in membrane domains rich in cholesterol and sphingolipids called cholesterol rafts or CRs (Morandat S 2002).
Different classes of lipid CRs coexist in a same cell. In addition to the classical CRs with no structural proteins, these ordered domains may be enriched in a structural protein component, which dramatically changes the morphology and function of the CR. This emerging protein class is called MORFs (modifiers of raft function). The first MORF identified was caveolin-1 (Cav-1). Three caveolin genes are known; caveolins 1 and 2 are ubiquitously expressed, whereas caveolin-3 is only expressed in astrocytes and muscle cells (Smart 1999).
Cav-1 is integrated into the CR microenvironment, but is also anchored to the cytoskeleton by its cytoplasmic domain. This arrangement allows CRs thus formed to generate dish-like 50-100 nm depressions (caveolae) and may be located or displaced in a regulated manner.
Although caveolae and CRs share certain biochemical properties, location of caveolins in caveolae differentiates those membrane domains. Since their discovery in the 50s (Yamaha 1955), there has been much speculation about the role of caveolins. Caveolae have been implied in multiple functions, including endocytosis, lipid homeostasis, tumorigenesis, calcium transport/regulation, cholesterol transport/regulation, and transcytosis of albumin and other proteins through endothelium (Simons 2000, Razani 2002). In addition, caveolins interact with proteins residing in caveolae. This protein-protein interaction involves, amongst others, the inducible nitric oxide synthetase (NOS 2) (Razani 2002).
The apical transport model based on CRs is based on lipid-lipid and lipid-protein relationships. It has recently been suggested that CRs could be important for transport through an endocytic pathway alternative to that of clathrin-coated vesicles. CRs could serve as an entry point for certain pathogens and toxins, such as Listeria monocytogenes (Seveau 2004). However, it had not been suggested that CRs could have any relevance for the transport of drugs known to cause cytotoxicity.
Imipenem is an antibiotic of the carbapenem class launched into the market in 1985. Imipenem is an antibacterial agent of the β-lactam class with a wide spectrum covering most Gram-negative and Gram-positive aerobic and anaerobic pathogens which has a marked activity against β-lactamase-producing species.
As other carbapenem antibiotics, imipenem undergoes a species-dependent extensive metabolism, as shown by the low recovery of active drug in urine (Birnbaum 1985, Kaham 1983). In vivo and in vitro studies showed that biotransformation mostly occurred in the kidney through the action of dehydropeptidase I (DHP-I). DHP-I hydrolyzes the β-lactam ring of imipenem and inactivates it (Kaham 1983). This phenomenon, called post-excretory metabolism, does not affect penicillins or cefalosporins.
The antibacterial spectrum of imipenem was suitable for treating Gram-negative germs, mainly in sepsis of an intestinal or renal origin. Renal degradation of imipenem caused suboptimal levels of active drug in the urinary tract (Kropp H 1982), thus limiting its use for the treatment of systemic infections. This caused preclinical research to be aimed at producing a potential inhibitor of DHP-I. This research program led to development of cilastatin, a compound structurally related to imipenem. The first reported studies in which cilastatin already appeared in combination with imipenem were presented to the 21st Interscience Conference on Antimicrobial Agents and Chemotherapy, held in Chicago in November 1981, by Norrby and colleagues (Norrby 1981).
Cilastatin is a competitive inhibitor of DHP-I that prevents hydrolysis of the peptide bond and opening of lactam rings. In the presence of cilastatin, dihydropeptidase does not open the lactam ring of imipenem, prevents its absorption, and increases urinary excretion of imipenem, reducing its concentration in tubular cells (Clissold 1987, Birnbaum 1985). High doses of imipenem alone may cause tubular toxicity in rabbits, but this effect is prevented by concomitant cilastatin administration (Norrby 1985).
In addition to its well known effect as renal dihydropeptidase inhibitor, cilastatin is able to inhibit organic anion transport systems (OATPs)
at basolateral level. This effect has recently been reported, and its implication in metabolism of other drugs has been discussed, with little success because the expected effect of its action upon OATPs would be to increase the circulating levels of the drugs involved and to decrease their overall clearance, exactly the opposite effects to those seen.
The chemical name of cilastatin sodium is the monosodium salt of [R-[R*,S*-(Z)]]-7-[(2-amino-2-carboxyethyl)thio]-2-[[(2,2-dimetylcyclopropyl) carbonyl]amino]-2-heptenoic acid. Its empirical formula is C16H25N2NaO5S, and it has a molecular weight of 380.44 Da. It is an off-white to yellowish white amorphous compound, hygroscopic, and highly soluble in water and methanol (Drusano G L 1984). Its chemical structure is as follows:

Cilastatin inhibits renal degradation of imipenem, achieving a urinary excretion rate up to 70% of the initial imipenem dose administered. An imipenem:cilastatin (I/C) ratio of 1:1 was established as the optimal dose for maintaining this inhibition for 8 to 10 hours (Norrby 1983). Multiple dose studies showed that cilastatin does not accumulate in healthy subjects.
Cilastatin was demonstrated to have a strong affinity for DHP-I by measuring levels of leukotriene E4, which is formed in the kidney from leukotriene D4 by renal dehydropeptidase in the brush border. In the presence of cilastatin, this conversion was strongly inhibited, which confirmed the cilastatin-DHP-I interaction (Koller 1985, Campbell 1988).
In the 80s, pharmacological research on cyclosporin A and its potential interactions with other commonly used drugs allowed for obtaining evidence that the I/C association could be useful for decreasing imipenem nephrotoxicity. Cyclosporin A (CsA) is a lipophilic cyclic endecapeptide, initially identified as an antifungal, for which a potent immunosuppressant activity was shown in 1972. It was finally approved for use in 1983. Introduction of CsA, alone or combined with other immunosuppressant agents, has markedly improved immunosuppressive therapy in solid organ and bone marrow transplants, and also in multiple autoimmune diseases. CsA mainly acts upon T cells by inhibiting the cascade of biochemical reactions highly dependent on calcium ion occurring after binding of an antigen to the T-cell receptor and which marks T-cell activation and proliferation through the synthesis of IL-2 (Belitsky 1986, Ryffel 1990).
The main side effect of CsA is nephrotoxicity, which affects both the native kidney (in cases of liver, heart, or bone marrow transplant) and the transplanted kidney (in kidney transplant) (Calne 1978). In both cases, the effect is dose-dependent. At tubular level, entry of CsA into the proximal tubule is very rapid. In vitro, 80% of the total is transported in the first 10 minutes at 30° C. (Jackson 1988). Morphological evidence suggests that sublethal tubular damage includes swelling of endoplasmic reticulum, isometric vacuolization, occurrence of autolysosomes, giant mitochondria, microcalcifications, changes in tubular epithelium cytoplasm, necrosis, sloughing, of tubular cells, tubular regeneration, and occasional increase in mitosis (Mihatsch 1986). Low CsA doses have been seen to initially cause a significant elevation in intracellular calcium levels before the loss of cell viability. CsA has also been shown to activate proapoptotic genes in tubular and interstitial cells.
However, inclusion of I/C in CsA treatment was seen to decrease renal function impairment in rats (Sido 1987). These experiments were confirmed in nephrectomized rats undergoing kidney transplant (Hammer 1989) and subsequently in humans. It was shown that cilastatin administration may effectively counteract the nephrotoxicity induced by CsA in the period immediately subsequent to a cardiac (Markewitz 1994), bone marrow (Gruss 1996), and kidney transplant (Carmellini 1997, 1998).
As cyclosporin A is not a substrate for DHP-I, the reasons for the nephroprotective effect of concomitantly administered imipenem/cilastatin (I/C) were not known to date, particularly because cilastatin alone was not available, and experiments in which cilastatin or imipenem were separately administered had therefore not been conducted. As a result, such effects could not clearly be attributed to one or the other drug. Mraz (1987, 1992) suggested that the nephroprotective effect, which he attributed to cilastatin, was due to reduction of plasma CsA concentrations. However, Markewitz found no significant differences in CsA doses or in blood levels of CsA or its metabolites, in agreement with Sido (Sido 1987) and Hammer (Hammer 1989). Gross noted that patients treated with I/C had lower CsA levels than patients not receiving I/C. However, the protective effect of I/C could not be attributed to changes in CsA levels (Gruss 1996).
In 1996, Toyoguchi showed that the I/C combination was able to reduce the nephrotoxicity of vancomycin in rabbits by inhibiting renal vancomycin accumulation (Toyoguchi 1996). One year later, while conducting studies on glomerular filtration of the animal, this author showed that cilastatin alone accelerated renal excretion of vancomycin, decreasing its levels in plasma and renal tissue (Toyoguchi 1997). Although his study included morphological data about vancomycin toxicity on renal tubule, the corresponding study with cilastatin was not provided. These results did not therefore suggest that the combination of both drugs could have clinical value, because an increase in renal excretion of vancomycin would theoretically decrease its effectiveness. Moreover, findings were not specific for the proximal tubule, because I/C decreased vancomycin concentration in both renal cortex and medulla.
Kusama et al conducted a pharmacokinetic study of the influence of the presence of cilastatin in whole animals administered vancomycin (Kusama 1998), and showed that the increase in vancomycin clearance induced by cilastatin was associated to a reduction in vancomycin renal clearance. These authors found no glomerular filtration changes with cilastatin, and could therefore not show any nephroprotective effect, but suggested the existence an potential effect, relating it to inhibition of dehydropeptidase 1 by cilastatin. They also suggested that cilastatin may have an effect on vancomycin reabsorption by the tubule, but as in studies by the Toyoguchi group, they did not conduct any direct study on the subject in cells, and could not therefore show the actual existence of such an effect. Although this group postulated that cilastatin competes with vancomycin for an entry point to the proximal cell, neither its studies nor the subsequent Nakamura studies (Nakamura 1998), in which the decrease in vancomycin excretion in the presence of imipenem/cilastatin was analyzed, allowed for concluding whether this was a competition with reabsorption or with tubular secretion of vancomycin (Nakamura 1998).
Neither CsA nor vancomycin are substrates for renal dipeptidase inhibitable by cilastatin. This is why the suggestions made in discussions of previous studies that a common transport system existed for imipenem and vancomycin met with no great enthusiasm, as it was usually considered that the mechanism by which this effect occurred was not elucidated. Thus, it was not evident for an expert in the technique that cilastatin could serve to decrease the nephrotoxicity of drugs other than those already mentioned. Although it has recently been reported that cilastatin is able to inhibit organic anion transport systems (OATPs) at basolateral level, hypotheses about its involvement in metabolism of other drugs have had little success, because the anticipated effect of its action on OATPs would be to increase circulating levels of the involved drugs and to reduce its global clearance. However, the effect usually seen is exactly the opposite. In fact, the brochure provided by the cilastatin manufacturer, Biomol International LP (brochure accessible in the Internet at http://www.biomol.com/SiteData/docs/ProductData/pi153.pdf), describes cilastatin as a dipeptidase inhibitor that inhibits hydrolysis of β-lactam antibiotics, attributing its nephroprotective action against the side effects of cyclosporin A to inhibition of dipeptidase of the brush border of the proximal tubule.
Cyclosporin is apolar, very sparingly soluble in water, and soluble in lipids and organic solutes, and the chance that it reaches the inside of cells of the proximal tubular epithelium using the organic anion transport system (OATP) is very low. Until now, its transport mechanism was considered to be transmembrane passive diffusion. Vancomycin has an amphoteric nature and is water-soluble, and could be a theoretical substrate for OATPs of the proximal tubule, but nobody has directly studied such possibility.
All in all, it was not obvious either for experts in the technique that there was any compound which could have an effect on the nephrotoxicity of multiple drugs, or even on nephrotoxic compounds with no known therapeutic activity, decreasing their harmful effects on the renal proximal tubule. This would however be of great interest, because nephrotoxicity is a problem that affects many drugs and limits administration of doses which would be required to achieve their effect on the condition intended to be treated. Nephrotoxic effects require dose reduction or drug discontinuation before full treatment has been administered. Therefore, identification of any compound that decreases the nephrotoxicity of a high number of drugs with nephrotoxic potential would be of great interest for clinical practice. The possibility of inhibiting a common entry mechanism would be a way to achieve this. This invention provides a solution to that problem.